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Metal binding kinetics of bi-histidine sites used in psi analysis: evidence of high-energy protein folding intermediates.

ABSTRACT: The zinc-specific fluorophore, Zinpyr-1, is used in competition assays to determine the kinetic and thermodynamic parameters of Zn2+ binding to engineered bi-histidine sites located in ubiquitin and the B domain of protein A (BdpA). These binding sites are used in psi analysis studies to investigate structure formation in the folding transition state identified by the change in folding rate upon addition of metal ions. For ubiquitin, the on-rate binding constant and binding affinity for a site located along an alpha-helix are measured to be approximately 10(7) M-1 s-1 and 3 microM, respectively. For a site located across two beta-strands, the metal binding affinity was too weak to measure in the dye competition assays (Kd > 55 microM). The equilibrium-determined values for the Zn2+-induced stabilization of ubiquitin and BdpA match the values derived from changes in the global folding and unfolding rates. Therefore, metal ion binding is in fast equilibrium during the transit over the free energy barrier. Accordingly, the folding rate must be slower than the product of the fractional population of a high-energy intermediate with the metal site formed and the metal binding on-rate constant. The known folding rate of 20 s-1 at 1.5 M guanidinium chloride in 400 microM Zn2+ provides an upper bound for the stability of such intermediates (DeltaG(U-I) < 4 kcal/mol). These results support a view of the apparent two-state protein folding reaction surface as a fast pre-equilibrium between the denatured state and a series of high-energy species. The net folding rate is a product of the equilibrium constant of the highest-energy species and a transmission rate. For ubiquitin, we estimate the transmission rate to be approximately 10(4) s-1. Implications for the role of unfolded chain diffusion on folding rates and barrier heights are discussed.

SUBMITTER: Bosco GL

PROVIDER: S-EPMC3313835 | biostudies-literature | 2009 Apr

REPOSITORIES: biostudies-literature

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Metal binding kinetics of bi-histidine sites used in psi analysis: evidence of high-energy protein folding intermediates.

Bosco Gerra L GL Baxa Michael M Sosnick Tobin R TR

Biochemistry 20090401 13

The zinc-specific fluorophore, Zinpyr-1, is used in competition assays to determine the kinetic and thermodynamic parameters of Zn2+ binding to engineered bi-histidine sites located in ubiquitin and the B domain of protein A (BdpA). These binding sites are used in psi analysis studies to investigate structure formation in the folding transition state identified by the change in folding rate upon addition of metal ions. For ubiquitin, the on-rate binding constant and binding affinity for a site l ...[more]

PMID: 19220047

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Project description:Determining the structure of the transition state is critical for elucidating the mechanism behind how proteins fold and unfold. Due to its high free energy, however, the transition state generally cannot be trapped and studied directly using traditional structural biology methods. Thus, characterizing the structure of the transition state that occurs as proteins fold and unfold remains a major challenge. Here, we report a novel (to our knowledge) method that uses engineered bi-histidine (bi-His) metal-binding sites to directly map the structure of the mechanical unfolding transition state of proteins. This method is adapted from the traditional ψ-value analysis, which uses engineered bi-His metal chelation sites to probe chemical (un)folding transition-state structure. The φ(M2+)(U)-value is defined as ΔΔG(‡-N)/ΔΔG(U-N), which is the energetic effects of metal chelation by the bi-His site on the unfolding energy barrier (ΔG(‡-N)) relative to its thermodynamic stability (ΔG(U-N)) and can be used to obtain information about the transition state in the mutational site. As a proof of principle, we used the small protein GB1 as a model system and set out to map its mechanical unfolding transition-state structure. Using single-molecule atomic force microscopy and spectrofluorimetry, we directly quantified the effect of divalent metal ion binding on the mechanical unfolding free energy and thermodynamic stability of GB1, which allowed us to quantify φ(M2+)(U)-values for different sites in GB1. Our results enabled us to map the structure of the mechanical unfolding transition state of GB1. Within GB1's mechanical unfolding transition state, the interface between force-bearing β-strands 1 and 4 is largely disrupted, and the first β-hairpin is partially disordered while the second β-hairpin and the α-helix remain structured. Our results demonstrate the unique application of ψ-value analysis in elucidating the structure of the transition state that occurs during the mechanical unfolding process, offering a potentially powerful new method for investigating the design of novel elastomeric proteins.

Dataset Information

Metal binding kinetics of bi-histidine sites used in psi analysis: evidence of high-energy protein folding intermediates.

Publications

Metal binding kinetics of bi-histidine sites used in psi analysis: evidence of high-energy protein folding intermediates.

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